Although inflammatory mechanisms underlying Inflammatory Bowel Disease and other organic gastrointestinal (GI) disorders have been identified, inflammatory pathways affecting functional gastrointestinal disorders and chronic GI symptoms with no identified organic cause, such as diarrhea and chronic abdominal pain (CAP), remain unclear. We found evidence for differential levels of specific candidate biomarkers in GI biopsies with histologically defined inflammatory phenotypes from patients without organic GI disorders. IL-6 levels in colonic biopsies from pediatric patients with inflammatory phenotypes were significantly increased compared to controls with non-inflammatory phenotypes (Henderson, et al. 2012, WJGP). In the same colonic biopsies, the mast cell numbers were significantly higher in patients with no evidence of histological inflammation compared to patients with inflammatory phenotypes. Interestingly, no difference in IL-6 and mast cell staining was observed in the upper GI tract biopsies, suggesting a significance of the colonic environment in chronic GI symptom pathology. Such significance was corroborated by our results using an intestinal permeability test solution, ingested by participants following an overnight fast. The test solution contains four sugar probes absorbable at various sites along the GI tract. Five hours after ingestion, excreted urine analysis of the four sugar probes and raffinose (an internal standard) was performed using mass spectrometry method. Based on these analyses, we showed that patients with CAP, meeting Rome III criteria for Irritable Bowel Syndrome (IBS), had a significantly decreased colonic permeability, represented by differential urinary sucralose output, compared to controls with no IBS. Of the IBS patients, 55% had diarrhea. Changes in sucrose urine output, representing gastric permeability, and changes in lactulose-to-mannitol ratio, representing small intestinal permeability, were not observed (Del Valle-Pinero et al., 2013). This unexpected finding, which points to altered colonic permeability as one of the likely mechanisms for the observed GI aberrations in patient with chronic GI symptoms, serves as a starting point for a closer investigation into candidate cellular and molecular players within the pathways and microenvironment contributing to chronic GI symptoms. Our preliminary investigation of such molecular players recently revealed mRNA and microRNA (miR) signatures associated with carefully phenotyped patients having chronic GI symptoms. In IBS patients, we found elevated levels of two circulating miRs, the hsa-miR-342-3p and hsa-miR-150, compared to controls with similar age, BMI, and ethnicity (Fourie et al., 2014), leading us to develop an epithelial-focused miR network in colorectal cell models (Joseph and Abey et al., 2018). Within the IBS group, IBS-constipation patients but not the IBS-diarrhea patients had elevated hsa-miR-342-3p level compared to controls. The involvement of inflammation is suggested by these specific miRs, which are linked to inflammation in other diseases, e.g., cancer. Other miR signatures were further found in a subset of patients with Non-alcoholic Fatty Liver Disease (NAFLD). Male NAFLD patients had significantly upregulated expressions of the hsa-miR-432-5p, hsa-miR-578 and hsa-miR-155-5p compared to female NAFLD patients. Some of these miRs are known to play a role in inflammatory mechanisms; thus, this finding suggests a gender-specific inflammatory miR signature in NAFLD(Longchamps et al., 2014). We also developed a network model of hypothetical co-regulatory motifs in a miRNA-mRNA interaction related to epithelial function (Robinson et al., 2018). In addition, we found candidate mRNA biomarkers which have been associated with inflammation: chemokine C-C motif ligand 16 (CCL-16) and the Vasoactive Intestinal Peptide (VIP), in IBS patients compared to controls. In subsets of IBS patients, the CCL-16 gene expression was also upregulated in IBS patients having chronic constipation, compared to both controls and IBS patients having chronic diarrhea (Del Valle Pinero et al., 2011). As per recent findings by others elucidating enhancement of CCL-16 protein function as a haptotactic gradient regulator for two key inflammatory cells, the monocytes and the lymphocytes, by matrix metalloproteases, our finding provides further evidence for a subclinical inflammatory mechanism in these patients. Likewise, the VIP gene expression from peripheral whole blood was significantly upregulated by 2.91-fold in IBS patients when compared to controls (Del Valle Pinero et al., 2015). VIP is being considered as a possible therapeutic target for several GI disorders, due to its response to inflammation in the GI tract and suppresses the immune system causing a reduction in the inflammation. To further evaluate findings of circulating miRs and mRNAs in patients with chronic GI dysfunction, we identified epithelial mucosa-adherent microbiome signatures in patients with IBS compared to controls (Fourie et al., 2016). We examined whether differences exist in the oral microbiome of IBS participants compared to healthy controls, and whether the oral microbiome relates to symptom severity. The oral buccal mucosal microbiome of 38 participants was characterized using PhyloChip microarrays. Participants reported severity of induced abdominal pain following ingestion of the previously utilized (Del Valle-Pinero et al., 2013) four-sugar probe intestinal permeability test solution. Induced abdominal pain post-ingestion was most severe in IBS participants, particularly those who were overweight, and was robustly correlated to the abundance of 60 OTUs, 4 genera, 5 families and 4 orders of bacteria. IBS-overweight participants showed decreased richness in the phylum Bacteroidetes and the genus Bacillus and a significant separation of the IBS-overweight group was evident through analysis of beta-diversity. Our oral microbial results are concordant with described fecal and colonic microbiome-IBS and -weight associations. Having IBS and being overweight, rather than IBS-subtypes (chronic constipation or diarrhea), was the most important factor in describing the severity of induced abdominal pain and variation in the microbiome. Further, pain severity strongly correlated to taxa-specific abundance, suggesting oral microbiomes' potential utilization in diagnosis and clinical phenotyping. We examined these microbiome results in conjunction with circulating molecular signatures (extracellular vesicles) in order to elucidate mechanisms underlying the observed altered colonic permeability in patients with chronic GI dysfunction (Abey et al., 2016). We utilize cell and animal models to investigate implications on gut epithelial barrier dynamics. Also, due to the increasingly recognized link between gut epithelial barrier dynamics and neurological function (Brain-Gut interactions), we examine the genetics of neuro-related behaviors, such as chronic stress and sleep, in our patients (Heitkemper et al., 2015; Reddy et al., 2014). Laboratory experiments designed to further explore these dynamics are in progress. We also explore the psychosocial implications of the biobehavioral effects of chronic abdominal pain and IBS. For instance, we found that individuals with IBS reported lower quality of life (Sherwin et al., 2017) and higher perceived stress compared to healthy controls. Harnessing shotgun proteomic analysis, we examined immunomodulatory mechanisms underlying IBS symptomatology and found differentially expressed proteins between participants with IBS and controls involving platelet-related associations in serotonergic signaling, inflammation, and immunomodulatory regulation. Further functional studies are underway.